Biological invasions are a major threat to biodiversity in the Neotropical region. However, few studies have evaluated the mechanisms underlying the long-term establishment of fish propagules in aquatic environments. Here, we associated fish biomass, species richness, and proportion of non-native species (contamination index) to quantify propagule and colonisation pressures, and fish biodiversity (measured by the Kempton’s index) in lakes and rivers of the Parana River floodplain. We organised species into native and non-native assemblages sampled by gillnetting and beach seining in spatio-temporal gradients, seasonally, from 2000 to 2017. Native and non-native Kempton’s indices were inversely correlated, native extinctions occurred locally with non-native biotic differentiation in lakes, rivers, and ecosystem contamination. A constant propagule pressure resulted in an overwhelming biodiversity of non-natives at the end of the evaluated time series. Biotic resistance to introduction was not evidenced in our deterministic trends. The observed patterns agreed with previous studies highlighting native biotic homogenisation and species extinctions, depending on biological invasions, landscape connectivity, and riverine impoundments. Long-term propagule pressure and non-native fish colonisation were the drivers of biodiversity that led to the predominance of non-native over native assemblages in the Parana River floodplain.